Mutant human factor IX with an increased resistance to inhibition by heparin

ABSTRACT

The present invention is related to a novel composition of matter and methods of using the same. More particularly, the invention describes mutant human factor IX which has an increased resistance to inhibition by heparin. Methods of making and using this composition for the therapeutic intervention of hemophilia are disclosed.

The present application is a U.S. National Phase Application ofinternational application no. PCT/US01/47276, which was filed Nov. 13,2001. That application claims benefit of priority of U.S. applicationSer. No. 60/248,326, which was filed Nov. 14, 2000, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides methods and compositions for use in thetreatment of blood coagulation disorders. More particularly, the presentinvention describes mutant human factor IX compositions for use in thetherapeutic intervention of hemophilia B.

BACKGROUND OF THE INVENTION

Hemophilia B (also known as Christmas disease) is one of the most commoninherited bleeding disorders in the world. It results in decreased invivo and in vitro blood clotting activity and requires extensive medicalmonitoring throughout the life of the affected individual. In theabsence of intervention, the afflicted individual may suffer fromspontaneous bleeding in the joints, which produces severe pain anddebilitating immobility; bleeding into muscles results in theaccumulation of blood in those tissues; spontaneous bleeding in thethroat and neck may cause asphyxiation if not immediately treated;bleeding into the urine; and severe bleeding following surgery, minoraccidental injuries, or dental extractions also are prevalent.

To the extent that the present invention relates to intervention ofblood clotting disorders, a brief discussion of the biological factorsand/or mechanisms involved in blood clotting is warranted. A blood clotis essentially a gelatinous mass, which seals blood vessels that havesustained an injury. Conversion of fluid blood to a blood clot involvesthe conversion of soluble fibrinogen, which is present in plasma, to theinsoluble gelatinous blood clot, composed primarily of cross-linkedfibrin. The conversion of fibrinogen to fibrin is the primary end resultof a multi-step process referred to as the blood coagulation cascade.This-cascade is a highly regulated process that involves the sequentialproteolytic conversion of serine proteases from zymogen to activeconformations, and subsequent formation of calcium-dependentphospholipid-bound enzyme complexes with specific protein cofactors.Normal in vivo blood coagulation at minimum requires the serineproteases factors II (prothrombin), VII, IX, X and XI (soluble plasmaproteins); cofactors including the transmembrane protein tissue factorand the plasma proteins factors V and VIII; fibrinogen, thetransglutaminase factor XIII, phospholipid (including activatedplatelets), and calcium. Additional proteins including kallikrein, highmolecular weight kininogen, and factor XII are required for some invitro clotting tests, and may play a role in vivo under pathologicconditions. The coagulation cascade is regulated by thethrombomodulin-protein C pathway, the fibrinolysis pathway, tissuefactor pathway inhibitor, and the serpin antithrombin III. Importantly,the inhibition of several coagulation proteases by antithrombin III(including factor IXa) is markedly accelerated by the anticoagulant drugheparin, as well as structurally similar heparan sulfate on theendothelial surface.

Upon injury, thrombocytes, in the presence of von Willebrand Factor (acomponent of clotting Factor VIII), cling to the collagen of injuredconnective tissue by adhesion. The thrombocytes change their form anddevelop protrusions, and in addition to this, their outer membranefacilitates the adhesion of further thrombocytes. Thereafter, varioussubstances are released from granula of these cells, which results invessel constriction as well as accumulation and activation of otherfactors of plasma blood clotting.

In hemophilia, blood clotting is disturbed by a lack of certain plasmablood clotting factors. Hemophilia B is caused by a deficiency in factorIX that may result from either the decreased synthesis of the factor IXprotein or a defective molecule with reduced activity. The treatment ofhemophilia occurs by replacement of the missing clotting factor byexogenous factor concentrates highly enriched in factor IX. However,generating such a concentrate is fraught with technical difficulties asdescribed below.

Factor IX, like other clotting factors, is naturally produced as aprecursor molecule having an additional pre-pro-sequence at theN-terminus. The pre-pro-sequence represents a signal sequence thatcauses the oriented transport of this protein in the cell. When thepre-pro Factor DC protein is secreted from the cell the pre-sequence iscleaved. The pro-sequence consists of about 15 to 18 amino acids andserves as a recognition sequence in carboxylation of glutamic acidresidues to 4-carboxy-L-glutamic acid. After successful carboxylation,the pro-sequence is also cleaved. If the pro-sequence is not cleaved oronly incompletely cleaved, only low activity clotting factors result.Human factor IX has a molecular weight of about 55,000 Dalton; when itspro-sequence is present the molecular weight is increased by about 2000Dalton.

Purification of factor IX from plasma almost exclusively yields activefactor IX. However, such purification of factor IX from plasma is verydifficult because factor IX is only present in low concentration inplasma [5 μg/mL; Andersson, Thrombosis Research 7: 451–459 (1975)].Efforts to produce recombinant factor IX have led to products with onlylow levels of activity [Kaufman et al., J. Biol Chem 261: 9622–9628(1986); Busby et al., Nature 316: 217–273 (1985); Rees et al., EMBO J 7:2053–2061 (1988)]. This can be traced back to an incomplete cleavage ofthe pro-sequence [Meulien et al., Prot Engineer 3: 629–633 (1990)]because a mixture of recombinantly produced pro-factor IX and factor IXis present in cell supernatants.

The in vivo activity of exogenously generated factor IX is limited bothby protein half-life and inhibitors of coagulation, includingantithrombin III. An additional factor that limits the use ofexogenously generated factor IX in an effective therapeutic protocol isthat endogenous heparan sulfate/heparin greatly inhibits the activity offactor IX that is used in the existing therapies for hemophilia B.

Heparin can inhibit factor IXa activity in the intrinsic tenase complex(factor IXa-factor VIIIa) directly, or markedly accelerate inhibition offactor IXa by antithrombin III. Heparan sulfate, a chemically similarglycosaminoglycan to heparin, is expressed widely in the body includingthe endothelial surface, where it has been demonstrated to accelerateinhibition of coagulation proteases by antithrombin III. Similarly, itmay directly inhibit intrinsic tenase activity at sites of injury,limiting the in vivo activity of factor IXa. Thus, a mutant factor IXathat is resistant to the effects of heparin/heparan sulfate and retainsin vitro clotting activity may have enhanced in vivo activity. Similarprotein engineering approaches have been used to enhance the therapeuticefficacy of other serine proteases, including improvement of the fibrinbinding properties of tissue plasminogen activator by mutagenesis.

Thus, there is a need for mutant factor IX, which has a reduced affinityfor heparin but retains it anti-clotting activity, and remains activewhen administered as part of a therapeutic regimen for hemophilia B.

SUMMARY OF THE INVENTION

The present invention provides novel mutant forms of factor IX that maybe used in the therapeutic intervention of hemophilia B. In a preferredembodiment, the present invention provides a mutant human factor IXcomprising a mutation in the heparin binding domain of factor IX, whichdecreases its affinity for heparin, as compared to wild-type humanfactor IX. By heparin binding domain, the present invention refers tothat site on the factor IX protein that binds to and interacts withheparin. In more specific embodiments, the mutation is a mutation of theamino acid residue 233, 230, 239, 241, 87, 91, 98, 101, or 92 ofwild-type human factor IX. In preferred embodiments, it is contemplatedthat one or more of these residues is mutated. In additionalembodiments, it is contemplated that the mutation further comprises asubstitution of arginine 170 of the wild-type human factor IX for analanine. The numbering system employed herein is the chymotrypsinnumbering system well known to those of skill in the art [Bajaj andBirktoft, Meth Enzymol 222:96–128 (1993)] and is also depicted herein inFIG. 3.

A preferred mutant human factor IX of the present invention has amutation of the amino acid located at residue number 233 of wild-typehuman factor IX, wherein said mutation decreases the affinity of saidmutant human factor IX for heparin as compared to wild-type human factorIX. More particularly, the mutation is a substitution of the arginine atposition 233 to any other amino acid. In still more specificembodiments, it is contemplated that the arginine at position 233 issubstituted with an alanine. Alternatively, the arginine at 233 or anyother arginine in the heparin binding domain is replaced with aglutamate.

Another aspect of the present invention describes a method of treating asubject having hemophilia comprising administering to said subject acomposition comprising a mutant human factor IX of the presentinvention, in an amount effective to promote blood clotting in saidsubject. Further methods contemplate administering to the subject acomposition comprising one or more additional blood clotting factorsother than said mutant human factor IX. In particular embodiments, thesubject suffering from hemophilia is suffering from hemophilia B.

Additional methods of treating hemophilia in a mammal also arecontemplated. Such methods comprise providing an expression constructcomprising a polynucleotide encoding a mutant factor IX of the presentinvention, operably linked to a promoter; and administering in a mammalan amount of the expression construct effective to allow the mutantfactor IX to be expressed at levels having a therapeutic effect on themammal. The indicia of a therapeutic effect of a given hemophiliatherapy are well known to those of skill in the art. For example, in thepresent invention, the therapeutic effect is an increased resistance offactor IX to inhibition by heparin. More particularly, the therapeuticeffect is a decrease in the blood clotting time of said mammal ascompared to the blood clotting time of said mammal in the absence ofsaid expression construct. In preferred aspects the expression constructcomprises a viral vector selected from the group consisting of anadenovirus, an adeno-associated virus, a retrovirus, a herpes virus, alentivirus and a cytomegalovirus. In preferred embodiments, theexpression control element is selected from the group consisting of acytomegalovirus immediate early promoter/enhancer, a skeletal muscleactin promoter and a muscle creatine kinase promoter/enhancer.

In specific embodiments, it is contemplated that the therapeuticcompositions (protein or expression vector compositions) of the presentinvention are administered to at least two sites in the mammal.

Yet another aspect of the present invention contemplates a recombinanthost cell stably transformed or transfected with a polynucleotideencoding a mutant human factor IX of the present invention in a mannerallowing the expression in the host cell of said mutant human factor IX.In specific embodiments, such a recombinant host cell may be employed inmethods of for the large scale production of the mutant human factor IXprotein.

Also contemplated by the present invention are pharmaceuticalcompositions comprising a mutant human factor IX protein of the presentinvention and a pharmaceutically acceptable carrier, excipient ordiluent. Also contemplated are pharmaceutical compositions comprising anexpression construct comprising a vector having an isolatedpolynucleotide encoding a mutant human factor IX of the instantinvention and a promoter operably linked to said polynucleotide; and apharmaceutically acceptable carrier, excipient or diluent. It iscontemplated that in addition to the mutant human factor IX, thepharmaceutical compositions may comprise additional therapeuticcomponents such as for example, other blood clotting factors andhemophilia therapeutic compositions.

Other aspects, features and advantages of the present invention will beapparent from the detailed description. It should be understood that thedetailed description presented below, while providing preferredembodiments of the invention, is intended to be illustrative only sincechanges and modification within the scope of the invention will bepossible whilst still providing an embodiment that is within the spiritof the invention as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate aspects of the present invention. Theinvention may be better understood by reference to one or more of thedrawings in combination with the detailed description of the specificembodiments presented herein.

FIG. 1. Purification of recombinant human factor IXa from 293 cells.Sliver stained nonreducing 10% SDS-PAGE representing samples frompurification steps. Lane 1 and 10-molecular weight markers, lane 2-conditioned media, lane 3 barium depleted media, lane 4-barium eluate,lane 5–9 fractions 17, 18, 19, 20, and 23 eluted from Mono Q HR 5/5 withcalcium chloride gradient (0–30 mM). The human factor IX isolated usingthis method demonstrated high purity by 10% SDS-PAGE with silverstaining, high specific clotting activity (187 U/mg), and an overallyield of approximately 30% by ELISA.

FIG. 2. Effect of unfractionated heparin on intrinsic tenase activityusing recombinant factor IXa in conditioned media. Serum-free mediaincubated for 48 hr following transient transfection of 293 cells wasconcentrated by Centricon-30 and activated with 2 nM human factor XIafor 2 hr at 37° C. The intrinsic tenase assay was performed in whichfactor Via was in excess (5 nM), and conditioned media served as theenzyme source. The rate of factor Xa generation in the presence ofincreasing amounts of unfractionated heparin is plotted for wild-type(●) and R233A (∘) factor IXa. Mock-transfected media demonstrated nosignificant activity. There was an increase in the residual activity inthe plateau phase for the mutant R233A (˜65%) relative to wild-typefactor IXa (˜15%).

FIG. 3A through FIG. 3C. Contiguous DNA (SEQ ID NO: 1) and amino acid(SEQ ID NO: 2) sequences of factor IX, including bold numbersunderneath, designating the amino acid sequence corresponding to thechymotrypsin numbering system.

FIG. 4A and FIG. 4B. The rate of factor Xa generation by 5 nM wild-type(●) or R233A (∘) factor IXa in the factor IXa-phospholipid (A) and theintrinsic tenase (B) complexes in the presence of increasing amounts ofunfractionated heparin. Tie mutant factor Ma R233A demonstratesincreased resistance to inhibition by heparin relative to wild-typefactor IXa.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hemophilia B is one of the most prevalent blood clotting disorders andresults from a deficiency of or defect in endogenous factor IX geneexpression or activity, respectively. Therapeutic intervention requiresreplacement therapy in which the patient is provided with exogenousfactor IX. However, treatment is limited by the commercial availabilityof clotting factor and the expense of treatment. Further, factor IX thatis isolated from natural sources or that is produced recombinantly usingnative sequences is inhibited by endogenous heparin/heparan sulfate inboth an antithrombin III-dependent and independent manner, limiting invivo activity and half-life of activated factor IXa. Hence the presentlyavailable replacement therapies are ineffective at providing an adequateremedy for the disease.

The present invention describes a mutant human factor IX that has anincreased resistance to heparin inhibition in vitro as compared towild-type human factor IX. More particularly, the invention describes amutant factor IX that has an Arg to Ala substitution at residue 233(according to the chymotrypsin numbering system, see FIG. 3, and SEQ IDNO: 2). The increased resistance of this mutant human factor IX toheparin means that the mutant human factor IX is a more effectivereplacement therapy for patients suffering from hemophilia B thanadministering wild-type factor IX. Further, it is expected that thismutant human factor IX also may possess an increased in vivo bloodclotting activity as compared to wild-type human factor IX. Methods andcompositions for exploiting the therapeutic potential of these findingsare discussed in further detail herein below.

A. Role of Factor IX in the Blood Coagulation Cascade.

Factor IX is a key serine protease that participates in the middle phaseof the blood clotting cascade. Factor IX is activated by either factorXIa or by factor VIIa-tissue factor in a Ca²⁺ dependent manner. Theactivated factor IXa with its cofactor VIIIa, in the presence of Ca²⁺and phospholipid, forms the intrinsic tenase complex and is responsiblefor generating activated Factor X. Factor IX is functionally deficientor absent in individuals with the inherited disorder hemophilia B.

Ex vivo modeling of blood coagulation demonstrates that formation of themembrane bound intrinsic tenase (factor IXa-factor VIIIa) andprothrombinase (factor Xa-factor Va) complexes results in a localized,explosive increase in thrombin generation [Lawson et al., J Biol Chem.,269(37):23357–66 (1994); Rand et al., Blood, 88(9):3432–45 (1996)]. Inminimally altered whole blood, the rate limiting factor for thrombingeneration is the activation of factor Xa by the intrinsic tenasecomplex [Rand et al., Blood, 88(9): 3432–45 (1996)]. In a cell-basedsystem containing platelets and monocytes expressing tissue factor,addition of picomolar factor IXa generates significantly more thrombinthan similar concentrations of factor Xa [Hoffman et al., Blood, 86(5):1794–801 (1995)]. Omitting either factor IX or factor VIII markedlyreduces the generation of thrombin [Lawson et al., J Biol Clem, 269(37):23357–66(1994)]. Thus, formation and activity of the intrinsic tenasecomplex is critical to the final rate of thrombin generation during thepropagation phase of coagulation. The activity of the intrinsic tenasecomplex appears to be primarily regulated by instability (loss of the A2domain) and proteolytic inactivation of factor VIIIa (by factor IXa)[Fay et al., J Biol Chem, 271(11): 6027–32 (1996)]. The pivotal role ofintrinsic tenase suggests that regulation of this enzyme complex iscritical to maintaining hemostatic balance.

Heparin has anticoagulant effects prolongation of in vitro coagulationassays) in plasma which are antithrombin (ATIII) dependent, largelyattributed to the acceleration of factor Xa and thrombin inhibition[Hirsh et al., Chest 108(4 Suppl) (1995)]. In both the purified stateand in plasma, ATIII inhibits factor IXa at a significantly slowerbaseline rate than thrombin and factor Xa [Jordan et al., J Biol Chem,255(21):10081–90 (1980); Pieters et al., J Biol Chem, 263(30):15313–8(1988); Pieters et al., Blood, 76(3): 549–54 (1990)]. However, additionof unfractionated heparin to contact-activated plasma results in asignificant increase in the amount of factor IXa-antithrombin complexformed [McNeely and Griffith, Blood, 65(5): 1226–31 (1985)]. Similar tothrombin, heparin acceleration of factor IXa inhibition by antithrombindemonstrates a biphasic dose response, and requires high molecularweight oligosaccharides for optimal rate enhancement [Holmer et al.,Biochem J, 193(2): 395–400 (1981); Mauray et al., Biochim Biophys Acta,1387(1–2): 184–94 (1998)]. These results suggest a template mechanismfor heparin catalysis, in which inhibition is accelerated by the bindingof protease and inhibitor to the same oligosaccharide chain.

In addition to accelerating protease inhibition by ATIII, heparin alsoinhibits the intrinsic tenase complex in an a ATIII independent manner[Barrow et al., J Biol Chem, 269(43): 26796–800 (1994)]. This inhibitionexhibits a partial, noncompetitive pattern, which is not explained byeffects on cofactor stability or assembly of the factor IXa-factor VIIIacomplex [Barrow et al., J Biol Chem, 269(43): 26796–800 (1994)]. Byeliminating potential effects on assembly or stability of the complexthat would reduce the effective enzyme concentration, these resultssuggest that heparin directly modulates the catalytic activity of theenzyme complex [Barrow et al., J Biol Chem, 269(43): 26796–800 (1994)].From mechanistic studies the inventor has inferred a model in whichheparin binds to a regulatory site on factor IX. The inventor has shownthat site directed mutagenesis of the heparin binding domain (atposition R233) generated a mutant human factor IX, which when comparedto wild-type factor IX, demonstrated markedly reduced inhibition byheparin. This mutant human factor IX as well as other mutant humanfactor IX proteins in which the regulatory site that binds heparin hasbeen disrupted will be useful in replacement therapy for individualssuffering from hemophilia.

The amino acid structure of human factor DC is well known to those ofskill in the art, see Bajaj and Birktoft [Meth Enzymol, 222: 96–128(1993)]. Given that the instant invention has shown that it is possibleto generate such a mutant, those of skill in the art will be able toproduce other mutants having a similar activity. Similarly, the nucleicacid sequence of the gene encoding factor IX also is well known to thoseof skill in the art (see FIG. 3 and SEQ ID NO: 1).

Of additional interest, recent studies have shown that the endocyticreceptor low density lipoprotein receptor-related protein (LRP) wasdemonstrated to bind factor IXa upon activation from a zymogen form in atwo-site binding model with equilibrium dissociation constants of 27 nMand 69 nM [eels et al., Blood 96(10): 3459–3465 (2000)]. Modification ofthe factor IXa active site, however, did not affect binding to LRP,suggesting that binding of factor IXa to LRP involves an enzyme exosite.LRP-deficient cells degrade 35% less factor IXa than LRP-expressingcells, suggesting a role for LRP in the transport of factor IXa to theintracellular degradation pathway. Degradation of factor IXa byproteoglycan-deficient cells proceeded at a rate lower than 83% thanthat of wild-type cells, also suggesting a role for proteoglycans in thebinding to LRP. Furthermore, the binding of factor IXa to LRP can befully inhibited in the presence of either 100 U/mL unfractionated or lowmolecular weight heparin. In contrast, little, if any, inhibition wasobserved in the presence of 100-μg/mL chondroitin sulfate. These dataindicate that the heparin-binding domain of factor IXa may contribute tothe interaction with LRP. Thus, factor IXa proteins with reducedaffinity for heparin may have reduced clearance by LRP-dependentmechanisms, further enhancing their in vivo activity.

B. Mutant Factor IX

The present invention contemplates the production of mutant humancoagulation factor IX that has an increased resistance to inhibition byheparin/heparan sulfate by both antithrombin-dependent and independentmechanisms. By mutant human factor IX, the present invention means humanfactor IX in which the wild-type sequence has been mutated.

Specifically contemplated by the present invention is site-specificmutagenesis of wild-type human factor IX. While the amino acid sequencevariants of the polypeptide can be substitutional mutants in which theamino acid at a given site is substituted for another, insertional ordeletion variants also are contemplated.

Substitutional variants typically exchange one amino acid of thewild-type for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptide, suchas stability against proteolytic cleavage, without the loss of otherfunctions or properties. Substitutions of amino acids to maintainactivity or properties preferably are conservative, that is, one aminoacid is replaced with one of similar shape and charge.

Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

Variant polypeptides include those wherein conservative substitutionshave been introduced by modification of polynucleotides encodingpolypeptides of the invention. Amino acids can be classified accordingto physical properties and contribution to secondary and tertiaryprotein structure. A conservative substitution is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are set out inthe Table A (from WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed Sep.6, 1996), immediately below.

TABLE A Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar G A P I L V Polar - uncharged C S T M N QPolar - charged D E K R Aromatic H F W Y Other N Q D EAlternatively, conservative amino acids can be grouped as described inLehninger. [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY(1975), pp.71–77] as set out in Table B, immediately below.

TABLE B Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D EAs still an another alternative, exemplary conservative substitutionsare set out in Table C, immediately below.

TABLE C Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

Also contemplated are non-conservative substitutions, in which an aminoacid is replaced with one of different properties. Replacement ofarginine or lysine to glutamate (charge reversal) to disrupt theelectrostatic binding of the protease to heparin, similar to thestrategy used for thrombin-heparin binding, is an example of suchnon-conservative substitutions [Sheehan and Sadler, Proc Nat'l Acad SciUSA, 91(12): 5518–22 (1994)]. Such nonconservative mutations may beuseful in generating further mutants that have increased resistance toheparin inhibition.

The binding of polyanionic heparin chains to factor IX(a) likelyinvolves interactions with basic amino acid residues on the proteasesurface. The binding of heparin to thrombin, a homologous coagulationprotease, is a highly electrostatic interaction that involves a numberof basic residues in exosite II [Sheehan and Sadler, Proc Nat'l Acad SciUSA, 91(12): 5518–22 (1994); Olson et al., J Biol Chem; 266(10): 6342–52(1991)]. The inventor prepared a three-dimensional structure of humanfactor Ixa by homology using SWISS-MODEL. Based on homology to thethrombin-heparin interaction, basic surface residues (lysine, arginine,or histidine) in the carboxyl-terminus -helix, and the insertion loop80–90 (chymotrypsin numbering) are appropriate targets for mutagenesis.Candidate residues include R87, H91, H92, K98, H101 in the 80–90 loopregion, and K230, R233, K239, and K241 in the carboxyl-terminus helix.It is expected that these mutations will be within the heparin bindingdomain of factor IX. Preferred mutants include single amino acidsubstitutions of alanine for R87, H92, R233, H101, and K241. Otherresidues are those that are within about 5Å that interact with theseaforementioned residues. The mutations may be combined with asubstitution of R170 to A170 [Chang et al., J Biol Chem, 273(20):12089–94 (1998)]. Of course these residues could also be mutated to anyother residue if desired, so long as the mutation provided a mutanthuman factor IX that was resistant to inhibition by heparin. In otherpreferred aspects, such a mutant human factor IX also retains bloodcoagulation activity. Using such mutagenesis also will allow mapping ofthe heparin binding site, similar to the mapping studies performed forthrombin [Sheehan and Sadler, Proc Nat'l Acad Sci USA, 91(12): 5518–22(1994)]. Further it is contemplated that mutations may be combined toprovide a more dramatic effect on heparin binding and function.

A preferred embodiment of the present invention contemplates generatinga mutation of the arginine at 233. This arginine may be mutated to anyamino acid. It should be noted that the mutants of the factor Ix peptideshould have an increased resistance to heparin inhibition. Such mutantsalso may preferably possess an increased clotting activity.

In order to construct mutants such as those described above, one ofskill in the art may employ well known standard technologies. Proteinsexpressed from such mutant can be assayed for appropriate heparininhibition and/or effect on blood clotting as described in furtherdetail below.

A random insertional mutation may also be performed by cutting the DNAsequence with a DNase I, for example, and inserting a stretch ofnucleotides that encode, 3, 6, 9, 12 etc., amino acids and religatingthe end. Once such a mutation is made the mutants can be screened forvarious activities presented by the wild-type protein.

Point mutagenesis also may be employed to identify with particularitythe amino acid residues that are important in particular activitiesassociated with the heparin binding of factor IX. Thus, one of skill inthe art will be able to generate single base changes in the DNA strandto result in an altered codon and a missense mutation.

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences that encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thenucleotide(s) being mutated. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to about 10 matchingbases on both sides of the nucleotide(s) being altered.

The technique typically employs a bacteriophage vector that exists inboth a single-stranded and double-stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double-strandedplasmids also are routinely employed in site-directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double-strandedvector, which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

A PCR-based method for site-directed mutagenesis is particularlypreferred. Overlapping forward (positive strand) and reverse (negativestrand) primers containing the desired mutation and 10–15 matchingnucleotides flanking both sides, are annealed with the denaturedwild-type cDNA in a suitable plasmid vector (i.e. Bluescript®). Thistemplate is then subject to amplification by PCR with a high fidelitythermostable DNA polymerase, the product digested with the restrictionendonuclease Dpn I (to degrade the methylated parental or wild-typeplasmid), and resulting DNA employed for transformation of bacteria.Antibiotic resistant bacterial colonies (containing the plasmid) arethen selected for overnight growth, isolation of plasmid DNA (miniprep),and sequencing to confirm the presence of the mutation.

C. Recombinant Protein Production.

Given the above disclosure of mutant human factor IX peptides it will bepossible for one of skill in the art to produce human factor IX peptidesby automated peptide synthesis, by recombinant techniques or both.

The mutant factor IX protein of the invention can be synthesized insolution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co., (1984); Tam et al., J Amer Chem Soc, 105: 6442 (1983);Merrifield, Science, 232: 341–347 (1986); and Barany and Merrifield, ThePeptides, Gross and Meienhofer, eds, Academic Press, New York, 1–284(1979), each incorporated herein by reference. The active protein can bereadily synthesized and then screened in screening assays designed toidentify reactive peptides.

Alternatively, a variety of expression vector/host systems may beutilized to contain and express a mutant factor IX coding sequence.These include but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid); or animal cell systems. Mammalian cells that are usefulin recombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Exemplary protocols for the recombinant expression of theprotein are described herein below.

A yeast system may be employed to generate the mutant peptides orproteins of the present invention. The coding region of the mutantfactor IX cDNA is amplified by PCR. A DNA encoding the yeastpre-pro-alpha leader sequence is amplified from yeast genomic DNA in aPCR reaction using one primer containing nucleotides 1–20 of the alphamating factor gene and another primer complementary to nucleotides255–235 of this gene [Kurjan and Herskowitz, Cell, 30: 933–943 (1982)].The pre-pro-alpha leader coding sequence and mutant factor IX codingsequence fragments are ligated into a plasmid containing the yeastalcohol dehydrogenase (ADH2) promoter, such that the promoter directsexpression of a fusion protein consisting of the pre-pro-alpha factorfused to the mature mutant factor IX polypeptide. As taught by Rose andBroach [Meth Enzymol, 185: 234–279, D. Goeddel, ed., Academic Press,Inc., San Diego, Calif. (1990)], the vector further includes an ADH2transcription terminator downstream of the cloning site, the yeast“2-micron” replication origin, the yeast leu-2d gene, the yeast REP1 andREP2 genes, the E. coli beta-lactamase gene, and an E. coli origin ofreplication. The beta-lactamase and leu-2d genes provide for selectionin bacteria and yeast, respectively. The leu-2d gene also facilitatesincreased copy number of the plasmid in yeast to induce higher levels ofexpression. The REP1 and REP2 genes encode proteins involved inregulation of the plasmid copy number.

The DNA construct described in the preceding paragraph is transformedinto yeast cells, using a known method, e.g., lithium acetate treatment[Stearns et al., Meth Enzymol, 185: 280–297 (1990)]. The ADH2 promoteris induced upon exhaustion of glucose in the growth media [Price et al.,Gene, 55: 287 (1987)]. The pre-pro-alpha sequence effects secretion ofthe fusion protein from the cells. Concomitantly, the yeast KEX2 proteincleaves the pre-pro sequence from the mature mutant factor IX [Bitter etal., Proc Nat'l Acad Sci USA, 81: 5330–5334 (1984)].

Alternatively, mutant factor IX may be recombinantly expressed in yeastusing a commercially available expression system, e.g., the PichiaExpression System (Invitrogen, San Diego, Calif.), following themanufacturer's instructions. This system also relies on thepre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOX1) promoter upon inductionby methanol.

The secreted mutant human factor IX is purified from the yeast growthmedium by, e.g., the methods used to purify mutant factor IX frombacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding mutant factor IX may be cloned into thebaculovirus expression vector pVL1393 (PharMingen, San Diego. Calif.).This mutant factor IX-containing vector is then used according to themanufacturer's directions (PharMingen) to infect Spodoptera frugiperdacells in sF9 protein-free media and to produce recombinant protein. Theprotein is purified and concentrated from the media using aheparin-Sepharose column (Pharmacia, Piscataway, N.J.) and sequentialmolecular sizing columns (Amicon, Beverly, Mass.), and resuspended inPBS. SDS-PAGE analysis shows a single band and confirms the size of theprotein, and Edman sequencing on a Porton 2090 Peptide Sequencerconfirms its N-terminal sequence.

Alternatively, the mutant factor IX may be expressed in an insectsystem. Insect systems for protein expression are well known to those ofskill in the art. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The mutantfactor IX coding sequence is cloned into a nonessential region of thevirus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of mutant factor IX willrender the polyhedrin gene inactive and produce recombinant viruslacking coat protein coat. The recombinant viruses are then used toinfect S. frugiperda cells or Trichoplusia larvae in which mutant factorIX is expressed [Smith et al., J Virol, 46: 584 (1983); Engelhard etal., Proc Nat'l Acad Sci USA, 91: 3224–7 (1994)].

In another example, the DNA sequence encoding the mature form of theprotein is amplified by PCR and cloned into an appropriate vector forexample, pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEX vector isdesigned to produce a fusion protein comprisingglutathione-S-transferase (GST), encoded by the vector, and a proteinencoded by a DNA fragment inserted into the vector's cloning site. Theprimers for the PCR may be generated to include, for example, anappropriate cleavage site.

The recombinant fusion protein may then be cleaved form the GST portionof the fusion protein. The pGEX-3X/mutant human factor IX construct istransformed into E. coli XL-1 Blue cells (Stratagene, La Jolla Calif.),and individual transformants were isolated and grown. Plasmid DNA fromindividual transformants is purified and partially sequenced using anautomated sequencer to confirm the presence of the desired mutant humanfactor IX encoding gene insert in the proper orientation.

While certain embodiments of the present invention contemplate producingthe mutant human factor IX protein using synthetic peptide synthesizersand subsequent FPLC analysis and appropriate refolding of the cysteinedouble bonds, it is contemplated that recombinant protein productionalso may be used to produce the mutant human factor IX peptidecompositions. For example, induction of the GST/mutant human factor IXfusion protein is achieved by growing the transformed XL-1 Blue cultureat 37° C. in LB medium (supplemented with carbenicillin) to an opticaldensity at wavelength 600 nm of 0.4, followed by further incubation for4 hours in the presence of 0.5 mM Isopropyl β-D-Thiogalactopyranoside(Sigma Chemical Co., St. Louis Mo.).

The fusion protein, expected to be produced as an insoluble inclusionbody in the bacteria, may be purified as follows. Cells are harvested bycentrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; andtreated with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15 minutes atroom temperature. The lysate is cleared by sonication, and cell debrisis pelleted by centrifugation for 10 minutes at 12,000 X g. The fusionprotein-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mMEDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000 Xg. The pellet is resuspended in standard phosphate buffered salinesolution (PBS) free of Mg⁺⁺ and Ca⁺⁺. The fusion protein is furtherpurified by fractionating the resuspended pellet in a denaturing SDSpolyacrylamide gel (Sambrook et al., supra). The gel is soaked in 0.4 MKCl to visualize the protein, which is excised and electroeluted ingel-running buffer lacking SDS. If the GST/mutant human factor IX fusionprotein is produced in bacteria as a soluble protein, it may be purifiedusing the GST Purification Module (Pharmacia Biotech).

The fusion protein may be subjected to digestion to cleave the GST fromthe mature mutant human factor IX protein. The digestion reaction (20–40μg fusion protein, 20–30 units human thrombin [4000 U/mg (Sigma) in 0.5mL PBS] is incubated 16–48 hrs, at room temperature and loaded on adenaturing SDS-PAGE gel to fractionate the reaction products. The gel issoaked in 0.4 M KCl to visualize the protein bands. The identity of theprotein band corresponding to the expected molecular weight of mutanthuman factor IX may be confirmed by partial amino acid sequence analysisusing an automated sequencer (Applied Biosystems Model 473A. FosterCity, Calif.).

Alternatively, the DNA sequence encoding the predicted mature mutanthuman factor IX protein may be cloned into a plasmid containing adesired promoter and, optionally, a leader sequence, see, for example,[Better et al., Science, 240: 1041–43 (1988)]. The sequence of thisconstruct may be confirmed by automated sequencing. The plasmid is thentransformed into E. coli strain MC1061 using standard proceduresemploying CaCl₂ incubation and heat shock treatment of the bacteria(Sambrook et al., supra). The transformed bacteria are grown in LBmedium supplemented with carbenicillin, and production of the expressedprotein is induced by growth in a suitable medium. If present, theleader sequence will affect secretion of the mature mutant human factorIX protein and be cleaved during secretion.

The secreted recombinant protein is purified from the bacterial culturemedia by the method described herein below.

Mammalian host systems for the expression of the recombinant proteinalso are well known to those of skill in the art. Host cell strains maybe chosen for a particular ability to process the expressed protein orproduce certain post-translation modifications that will be useful inproviding protein activity. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing, which cleaves a “prepro” form of theprotein, may also be important to correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, andthe like have specific cellular machinery and characteristic mechanismsfor such post-translational activities and may be chosen to ensure thecorrect modification and processing of the introduced, foreign protein.

In a particularly preferred method of recombinant expression of themutant human factor IX proteins of the present invention 293 cells areco-transfected with plasmids containing the mutant human factor IX cDNAin the pCMV vector (5′ CMV promoter, 3′ HGH poly A sequence) and pSV2neo(containing the neo resistance gene) by the calcium phosphate method.Preferably, the vectors should be linearized with ScaI prior totransfection. Similarly an alternative construct using a similar pCMVvector with the neo gene incorporated can be used. Stable cell lines areselected from single cell clones by limiting dilution in growth mediacontaining 0.5 mg/mL G418 (neomycin like antibiotic) for 10–14 days.Cell lines are screened for mutant factor IX expression by ELISA orWestern blot, and high expressing cell lines are expanded for largescale growth.

It is preferable that the transformed cells are used for long-term,high-yield protein production and as such stable expression isdesirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellsmay be allowed to grow for 1–2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside G418; also that confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

D. Protein Purification

It will be desirable to purify the mutant factor IX proteins generatedby the present invention. Protein purification techniques are well knownto those of skill in the art. These techniques involve, at one level,the crude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; and isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE [Capaldi et al.,Biochem Biophys Res Comm, 76: 425 (1977)]. It will therefore beappreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified expressionproducts may vary.

In particular, the present invention incorporates herein by referenceU.S. Pat. No. 6,063,909; U.S. Pat. No. 6,034,222; U.S. Pat. No.5,639,857 (each incorporated herein by reference). These documentsdescribe specific exemplary methods for the isolation and purificationof factor IX compositions that may be useful in isolating and purifyingthe mutant human factor IX of the present invention. Given thedisclosure of these patents, it is evident that one of skill in the artwould be well aware of numerous purification techniques that may be usedto purify factor IX from a given source.

U.S. Pat. No. 6,063,909 provides methods and compositions for protectingblood coagulation factor IX from proteases during purification orstorage. Such methods employ high concentrations of one or more watersoluble organic or inorganic salts to stabilize factor IX againstconversion to clinically unacceptable peptide structures such as factorIXa, and/or degraded factor IX peptides. The technique is useful instabilizing intermediate purity factor IX preparations duringpurification, and in maintaining the integrity of purified factor IXduring long term storage. One of skill in the art may use methods suchas those disclosed in U.S. Pat. No. 6,063,909 in combination with theinstant invention to provide additional stability to the factor IXpreparations of the present invention.

U.S. Pat. No. 6,034,222 describes a method for the chromatographicseparation of recombinant pro-factor IX from recombinant factor IX,which employs ion exchangers such as QAE (QAE-Sephadex®, a strong basicanion exchanger comprised of dextran gels that are modified byintroduction of N,N-diethyl-N-(2-hydroxy-1-propyl)-ammonio-ethylgroups), DEAE (DEAE cellulose, diethylaminoethyl cellulose, anionexchanger) or TMAE (TMAE cellulose, triethylammonioethyl cellulose) andsubsequent elution of factor IX by buffer solutions with high saltconcentrations and/or low pH values.

Yet another method for the purification of mutant factor IX contemplatesthe use of immunoaffinity chromatography using an immunoadsorbentcomprising a monoclonal antibody. See, for example, [Liebman et al.Blood, 62(5), supp. 1, 288a (1983); Liebman et al., Proc Nat'l Acad SciUSA, 82: 3879–3883 (1985); Bessos, Thrombosis and Haemostasis, 56(1):86–89 (1986)]. U.S. Pat. No. 5,614,500 describes an immunoaffinitypurification of factor IX conducted in the presence of a chelatingagent. The techniques described therein may be useful in the presentinvention.

Also it is contemplated that a combination of anion exchange andimmunoaffinity chromatography may be employed to produce purified mutantfactor IX compositions of the present invention.

In a particularly preferred protocol for protein purification, serumfree media containing 10 μg/mL Vitamin K is incubated with a confluentcell line expressing the mutant human factor IX protein, and harvestedevery 48 hrs for 10 days. Benzamidine (5 mM) is added, the mediacentrifuged at 1200 g to eliminate cellular and particulate debris, andthe conditioned media frozen at −25° C. Upon thawing, the conditionedmedia is pooled, filtered, and subjected to barium chlorideprecipitation [Cote et al., J Biol Chem, 269(15): 11374–80 (1994)]. Theprecipitate is dissolved in 0.2 M EDTA, and the eluate dialyzedovernight before application to a Mono Q HR 5/5 column(0.15 M NaCl, 20mM HEPES, pH 7.4, 0.1% PEG-8000). Human factor IX is eluted with acalcium chloride gradient (0–45 mM) and concentrated in a Centricon-30.This approach selects for fully gamma-carboxylated factor IX based onthe specificity of the calcium chloride elution. Since this purificationtakes advantage of the unique properties of the Gla domain, mutationsintroduced into the protease domain are not expected to affectpurification of the proteins.

E. Vectors for Cloning, Gene Transfer and Expression.

As discussed in the previous section, expression vectors are employed toexpress the mutant human factor IX polypeptide product, which can thenbe purified and used in replacement therapy for the treatment ofhemophilia B. In other embodiments, expression vectors may be used ingene therapy applications to introduce the mutant factor IX-encodingnucleic acids into cells in need thereof and/or to induce mutant factorIX expression in such cells. The present section is directed to adescription of the production of such expression vectors.

Expression requires that appropriate signals be provided in the vectors,and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare described. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products also are provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

a. Regulatory Elements.

Promoters and Enhancers. Throughout this application, the term“expression construct” or “expression vector” is meant to include anytype of genetic construct containing a nucleic acid coding for geneproducts in which part or all of the nucleic acid encoding sequence iscapable of being transcribed. The transcript may be translated into aprotein, but it need not be In certain embodiments, expression includesboth transcription of a gene and translation of mRNA into a geneproduct. The nucleic acid encoding a gene product is undertranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7–20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30–110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, β-actin, rat insulin promoter, the phosphoglycerolkinase promoter and glyceraldehyde-3-phosphate dehydrogenase promoter,all of which are promoters well known and readily available to those ofskill in the art, can be used to obtain high-level expression of thecoding sequence of interest. The use of other viral or mammaliancellular or bacterial phage promoters that are well-known in the art toachieve expression of a coding sequence of interest is contemplated aswell, provided that the levels of expression are sufficient for a givenpurpose. By employing a promoter with well known properties, the leveland pattern of expression of the protein of interest followingtransfection or transformation can be optimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. Several inducible promoter systems are available forproduction of viral vectors. One such system is the ecdysone system(Invitrogen, Carlsbad. Calif.), which is designed to allow regulatedexpression of a gene of interest in mammalian cells. It consists of atightly regulated expression mechanism that allows virtually no basallevel expression of the transgene, but over 200-fold inducibility.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, Calif.) originally developed by Gossen andBujard [Proc Nat'l Acad Sci USA, 15;89(12):5547–51 (1992); Gossen etal., Science, 268(5218): 1766–69 (1995)].

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter is often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoetic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

Similarly, tissue specific promoters may be used to affect transcriptionin specific tissues or cells to reduce potential toxicity or undesirableeffects on non-targeted tissues. For example, promoters such as the PSA,probasin, prostatic acid phosphatase, or prostate-specific glandularkallikrein (hK2) may be used to target gene expression in the prostate.

In certain indications, it may be desirable to activate transcription atspecific times after administration of the gene therapy vector. This maybe done with such promoters as those that are hormone or cytokineregulatable. For example, in gene therapy applications where theindication is a gonadal tissue where specific steroids are produced orrouted to, use of androgen or estrogen regulated promoters may beadvantageous. Such promoters that are hormone regulatable include MMTV.MT-1, ecdysone, and RuBisco. Other hormone regulated promoters such asthose responsive to thyroid, pituitary, and adrenal hormones areexpected to be useful in the present invention. Cytokine andinflammatory protein responsive promoters that could be used include Kand T Kininogen [Kageyama et al., J Biol Chem, 262(5): 2345–51 (1987)],c-fos, TNF-alpha, C-reactive protein [Arcone et al., Nucl Acids Res16(8): 3195–207 (1988)], haptoglobin [Oliviero et al., EMBO J, 6(7):1905–12 (1987)], serum amyloid A2, C/EBP alpha, IL-1, IL-6 [Poli andCortese, Proc Nat'l Acad Sci USA, 86(21): 8202–6 (1989)], complement C3[Wilson et al., Mol Cell Biol 10(12): 6181–91 (1990)], IL-8, alpha-1acid glycoprotein [Prowse and Baumann, Mol Cell Biol, 8(1): 42–51(1988)], alpha-1 antitypsin, lipoprotein lipase [Zechner et al., MolCell Biol, 8(6): 2394–401 (1988)], angiotensinogen [Ron et al., Mol CellBiol 11(5): 2887–95 (1991)], fibrinogen, c-jun (inducible by phorbolesters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide),collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin, and alpha-1 antichymotrypsin.

Other promoters that could be used according to the present inventioninclude Lac-regulatable, heat (hyperthermia)-inducible promoters, andradiation-inducible, for e.g., EGR [Joki et al., Hum Gene Ther; 6(12):1507–13 (1995)], alpha-inhibin, RNA pol III tRNA met and other aminoacid promoters. U1 snRNA [Bartlett et al., Proc Nat'l Acad Sci USA,20;93(17): 8852–7 (1996)], MC-1, PGK, β-actin, and α-globin. Many otherpromoters that may be useful are listed in Walther and Stein [J Mol Med74(7): 379–92 (1996)].

It is envisioned that any of the above promoters alone, or incombination with another, may be useful according to the presentinvention depending on the action desired. In addition, this list ofpromoters should not be construed to be exhaustive or limiting, andthose of skill in the art will know of other promoters that may be usedin conjunction with the promoters and methods disclosed herein.

Another regulatory element contemplated for use in the present inventionis an enhancer. These are genetic elements that increase transcriptionfrom a promoter located at a distant position on the same molecule ofDNA. Enhancers are organized much like promoters. That is, they arecomposed of many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization. Enhancers useful in the present invention are well knownto those of skill in the art and will depend on the particularexpression system being employed [Scharf et al., Results Probl CellDiffer 20: 125–62 (1994); Bittner et al., Meth Enzymol 153: 516–544(1987)].

Polyadenylation Signals. Where a cDNA insert is employed, one willtypically desire to include a polyadenylation signal to affect properpolyadenylation of the gene transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and any such sequence may be employed, suchas human or bovine growth hormone and SV40 polyadenylation signals. Alsocontemplated as an element of the expression cassette is a terminator.These elements can serve to enhance message levels and to minimizeread-through from the cassette into other sequences.

IRES. In certain embodiments of the invention, the use of internalribosome entry site (IRES) elements is contemplated to create multigene,or polycistronic, messages. IRES elements are able to bypass theribosome scanning model of 5′ methylated Cap dependent translation andbegin translation at internal sites [Pelletier and Sonenberg, Nature,334: 320–325 (1988)]. IRES elements from two members of the picornavirusfamily (poliovirus and encephalomyocarditis) have been described[Pelletier and Sonenberg (1988), supra], as well an IRES from amammalian message [Macejak and Sarnow, Nature, 353: 90–94 (1991)]. IRESelements can be linked to heterologous open reading frames. Multipleopen reading frames can be transcribed together, each separated by anIRES, creating polycistronic messages. By virtue of the IRES element,each open reading frame is accessible to ribosomes for efficienttranslation. Multiple genes can be efficiently expressed using a singlepromoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

b. Delivery of Expression Vectors.

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. In other embodiments, non-viral delivery is contemplated. Theability of certain viruses to enter cells via receptor-mediatedendocytosis, to integrate into host cell genome and express viral genesstably and efficiently have made them attractive candidates for thetransfer of foreign genes into mammalian cells [Ridgeway, In: RodriguezR L, Denhardt D T, ed. Vectors: A survey of molecular cloning vectorsand their uses. Stoneham: Butterworth, 467–492, 1988; Nicolas andRubenstein, In: Vectors: A survey of molecular cloning vectors and theiruses, Rodriguez & Denhardt (eds.), Stoneham: Butterworth, 493–513, 1988;Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York,Plenum Press, 117–148, 1986; Temin, In: gene Transfer, Kucherlapati(ed.), New York: Plenum Press, 149–188, 1986]. The first viruses used asgene vectors were DNA viruses including the papovaviruses (simian virus40, bovine papilloma virus, and polyoma) [Ridgeway, (1988), supra;Baichwal and Sugden, (1986), supra] and adenoviruses [Ridgeway, (1988),supra; Baichwal and Sugden, (1986), supra]. These have a relatively lowcapacity for foreign DNA sequences and have a restricted host spectrum.Furthermore, their oncogenic potential and cytopathic effects inpermissive cells raise safety concerns. They can accommodate only up to8 kb of foreign genetic material but can be readily introduced in avariety of cell lines and laboratory animals [Nicolas and Rubenstein,(1988), supra; Temin, (1986), supra].

It is now widely recognized that DNA may be introduced into a cell usinga variety of viral vectors. In such embodiments, expression constructscomprising viral vectors containing the genes of interest may beadenoviral (see, for example, U.S. Pat. No. 5,824,544; U.S. Pat. No.5,707,618; U.S. Pat. No. 5,693,509; U.S. Pat. No. 5,670,488; U.S. Pat.No. 5,585,362; each incorporated herein by reference), retroviral (see,for example, U.S. Pat. No. 5,888,502; U.S. Pat. No. 5,830,725; U.S. Pat.No. 5,770,414; U.S. Pat. No. 5,686,278; U.S. Pat. No. 4,861,719 eachincorporated herein bad reference), adeno-associated viral (see, forexample, U.S. Pat. No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No.5,622,856; U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat.No. 5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S.Pat. No. 5,851,521; U.S. Pat. No. 5,252,479 each incorporated herein byreference), an adenoviral-adenoassociated viral hybrid (see, forexample, U.S. Pat. No. 5,856,152 incorporated herein by reference) or avaccinia viral or a herpesviral (see, for example, U.S. Pat. No.5,879,934; U.S. Pat. No. 5,849,571; U.S. Pat. No. 5,830,727; U.S. Pat.No. 5,661,033; U.S. Pat. No. 5,328,688 each incorporated herein byreference) vector.

There are a number of alternatives to viral transfer of geneticconstructs. This section provides a discussion of methods andcompositions of non-viral gene transfer. DNA constructs of the presentinvention are generally delivered to a cell, and in certain situations,the nucleic acid or the protein to be transferred may be transferredusing non-viral methods.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells are contemplated by the present invention.These include calcium phosphate precipitation [Graham and Van Der Eb,Virology, 52: 456–467 (1973); Chen and Okayama, Mol Cell Biol, 7:2745–2752 (1987); Rippe et al., Mol Cell Biol, 10: 689–695 (1990)]DEAE-dextran [Gopal, Mol Cell Biol, 5: 1188–1190 (1985)],electroporation [Tur-Kaspa et al., Mol Cell Biol, 6: 716–718 (1986);Potter et al., Proc Nat'l Acad Sci USA, 81: 7161–7165 (1984)], directmicroinjection [Harland and Weintraub, J Cell Biol, 101: 1094–1099(1985)], DNA-loaded liposomes [Nicolau and Sene, Biochim Biophys Acta,721: 185–190 (1982); Fraley et al., Proc Nat'l Acad Sci USA, 76:3348–3352 (1979); Felgner, Sci Amer 276(6): 102–6 (1997); Feigner, HumGene Ther 7(15): 1791–3 (1996)], cell sonication [Fechheimer et al.,Proc Nat'l Acad Sci USA, 84: 8463–8467 (1987)], gene bombardment usinghigh velocity microprojectiles [Yang et al., Proc Nat'l Acad Sci USA,87: 9568–9572 (1990)], and receptor-mediated transfection [Wu and Wu, JBiol Chem, 262: 4429–4432 (1987); Wu and Wu, Biochemistry, 27: 887–892(1988); Wu and Wu, Adv Drug Deliv Rev, 12: 159–167 (1993)].

Once the construct has been delivered into the cell, the nucleic acidencoding the therapeutic gene may be positioned and expressed atdifferent sites. In certain embodiments, the nucleic acid encoding thetherapeutic gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell, and where inthe cell the nucleic acid remains, is dependent on the type ofexpression construct employed.

In a particular embodiment of the invention, the expression constructmay be entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers [Ghosh andBachhawat, In: Liver diseases, targeted diagnosis and therapy usingspecific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker,pp. 87–104, (1991)]. The addition of DNA to cationic liposomes causes atopological transition from liposomes to optically birefringentliquid-crystalline condensed globules [Radler et al., Science,275(5301): 810–4 (1997)]. These DNA-lipid complexes are potentialnon-viral vectors for use in gene therapy and delivery.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Also contemplated in the presentinvention are various commercial approaches involving “lipofection”technology. In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA [Kaneda et al., Science, 243: 375–378 (1989)].In other embodiments, the liposome maybe complexed or employed inconjunction with nuclear nonhistone chromosomal proteins (HMG-1) [Katoet al., J Biol Chem, 266: 3361–3364 (1991)]. In yet further embodiments,the liposome may be complexed or employed in conjunction with both HVJand HMG-1. In that such expression constructs have been successfullyemployed in transfer and expression of nucleic acid in vitro and invivo, then they are applicable for the present invention.

Other vector delivery systems that can be employed to deliver a nucleicacid encoding a therapeutic gene into cells include receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific [Wu and Wu, (1993),supra].

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) [Wuand Wu, (1987), supra] and transferrin [Wagner et al., Proc Nat'l AcadSci USA, 87(9): 3410–3414 (1990)]. Recently, a syntheticneoglycoprotein, which recognizes the same receptor as ASOR, has beenused as a gene delivery vehicle [Ferkol et al., FASEB J. 7: 1081–1091(1993); Perales et al., Proc Nat'l Acad Sci USA, 91: 4086–4090 (1994)]and epidermal growth factor (EGF) has also been used to deliver genes tosquamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. [Meth Enzymol, 149: 157–176(1987)] employed lactosyl-ceramide, a galactose-terminalasialganglioside, incorporated into liposomes and observed an increasein the uptake of the insulin gene by hepatocytes. Thus, it is feasiblethat a nucleic, acid encoding a therapeutic gene also may bespecifically delivered into a particular cell type by any number ofreceptor-ligand systems with or without liposomes.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be preformed by any of the methods mentioned above thatphysically or chemically permeabilize the cell membrane. This isapplicable particularly for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky et al. [Proc Nat'l Acad SciUSA, 81: 7529–7533 (1984)] successfully injected polyomavirus DNA in theform of CaPO₄ precipitates into liver and spleen of adult and newbornmice demonstrating active viral replication and acute infection.Benvenisty and Neshif [Proc Nat'l Acad Sci USA, 83:9551–9555 (1986)]also demonstrated that direct intraperitoneal injection of CaPO₄precipitated plasmids results in expression of the transfected genes.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them [Klein et al., Nature, 327: 70–73 (1987)].Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force [Yang etal., Proc Nat'l Acad Sci USA, 87: 9568–9572 (1990)]. Themicroprojectiles used have consisted of biologically inert substancessuch as tungsten or gold beads.

F. Methods of Treating Hemophilia B.

As mentioned herein above, it is contemplated that the mutant humanfactor IX protein or the vectors comprising a polynucleotide encodingsuch a protein will be employed in replacement therapy protocols for thetreatment of hemophilia B.

a. Protein Based Therapy

One of the therapeutic embodiments of the present invention is theprovision, to a subject in need thereof, compositions comprising themutant human factor IX protein of the present invention. As discussedabove, the protein may have been generated through recombinant means orby automated peptide synthesis. The factor IX formulations for such atherapy may be selected based on the route of administration and mayinclude liposomal formulations as well as classic pharmaceuticalpreparations.

The mutant human factor IX proteins are formulated into appropriatepreparation and administered to one or more sites within the subject ina therapeutically effective amount. In particularly preferredembodiments, the mutant human factor IX protein based therapy iseffected via continuous or intermittent intravenous administration. By“therapeutically effective amount” the present invention refers to thatamount of mutant human factor IX that is sufficient to produce orenhance the coagulation of blood in a mammal following a bleed. Forexample, a therapeutically effective amount may enhance coagulation byreducing clotting times in a blood coagulation assay, or even increaseformation of intrinsic tenase or factor X activation. Blood coagulationassays are well known to those of skill in the art and are described forexample, in Walter et al., [Proc Nat'l Acad Sci USA, 93: 3056–3061(1996); Hathaway and Goodnight (1993), Laboratory Measurement ofHemostasis and Thrombosis, In: Disorders of Hemostasis and Thrombosis: AClinical Guide, pp. 21–29)].

Those of skill in the art will understand that the amounts of mutanthuman factor IX for therapeutic use may vary. It is contemplated thatthe specific activity of the factor IX protein preparation may be in therange of from about 100 units/mg of protein to about 500 units/mgprotein. Thus, a given preparation of mutant human factor IX maycomprises about 100 units/mg protein, about 125 units/mg protein, about150 units/mg protein, about 175 units/mg protein, about 200 units/mgprotein, about 225 units/mg protein, about 250 units/mg protein, about275 units/mg protein, about 300 units/mg protein, about 325 units/mgprotein, about 350 units/mg protein, about 375 units/mg protein, about400 units,/mg protein, about 425 units/mg protein, about 450 units/mgprotein, about 475 units/mg protein and about 500 units/mg protein. Aparticularly preferred range is from about 100 units/mg protein to about200 units/mg protein, a more preferable range is between about 150 toabout 200 units/mg protein. Preferably, the protein composition issubstantially free of contaminating factor IXa and has a factor IXacontamination level of less than 0.02% (w/w). Factor IX compositions,suitable for injection into a patient, can be prepared for example, byreconstitution with a pharmacologically acceptable diluent of alyophilized sample comprising purified factor IX and stabilizing salts.

Administration of the compositions can be systemic or local and maycomprise a single-site injection of a therapeutically effective amountof the mutant human factor IX protein composition. Any route known tothose of skill in the art for the administration of a therapeuticcomposition of the invention is contemplated including for example,intravenous, intramuscular, subcutaneous, or a catheter for long-termadministration. Alternatively, it is contemplated that the therapeuticcomposition may be delivered to the patient at multiple sites. Themultiple administrations may be rendered simultaneously or may beadministered over a period of several hours. In certain cases it may bebeneficial to provide a continuous flow of the therapeutic composition.Additional therapy may be administered on a periodic basis, for example,daily, weekly, or monthly.

b. Genetic Based Therapies.

Another therapeutic embodiment contemplated by the present invention isa method of treating a mammal having hemophilia comprising administeringto the mammal a gene therapy based pharmaceutical composition.Specifically, the present inventors intend to provide, to a given tissuein a patient or subject in need thereof, an expression construct capableof providing the mutant human factor IX to that patient in a functionalform. It is specifically contemplated that a gene encoding the mutanthuman factor IX will be employed in human therapy. The lengthydiscussion of expression vectors and the genetic elements employedtherein is incorporated into this section by reference. Particularlypreferred expression vectors are viral vectors such as adenovirus,adeno-associated virus, herpesvirus, vaccinia virus, and retrovirus.Also preferred is a liposomally-encapsulated expression vector.

Those of skill in the art are well aware of how to apply gene deliveryin vivo. For viral vectors, one generally will prepare a viral vectorstock. Depending on the kind of virus and the titer attainable, one willdeliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹ or1×10¹² infectious particles to the patient. Similar figures may beextrapolated for liposomal or other non-viral formulations by comparingrelative uptake efficiencies. Formulation as a pharmaceuticallyacceptable composition is discussed below.

Various routes are contemplated for delivery. The section below onroutes contains an extensive list of possible routes. For example,systemic delivery is contemplated. In other cases, a variety of direct,local and regional approaches may be taken. For example, where theindividual being treated exhibits a localized bleed, that area may bedirectly injected with the expression vector.

In certain embodiments, it is contemplated that a preparation of thevector comprising the mutant human factor IX encoding polynucleotide isinjected into the muscle tissue of an animal at a single site per dose.In other embodiments, the preparation is injected into the muscle tissueof the animal either simultaneously, or over the course of severalhours, at multiple muscle tissue sites. In the latter instance, when themethod comprises simultaneous multiple injections of viral vectorgenomes, it is envisaged that a multiple delivery injection device maybe used such that different areas of muscle tissue receive the vectorsimultaneously.

Incorporated herein by reference is U.S. Pat. No. 6,093,392 thatdescribes methods of gene therapy for hemophilia, which employadeno-associated viral vectors. Similarly, U.S. Pat. No. 5,935,935 isincorporated herein by reference and describes the use of adenoviralvectors for the treatment of hemophilia. It is contemplated that themethods described therein will be useful in combination with thecompositions of the present invention.

Also incorporated herein by reference is U.S. Pat. No. 5,681,746, whichdescribes retroviral vectors for the expression of factor VIII andpharmaceutical compositions and methods of using such vectors fortreating hemophilia. The present invention contemplates gene therapyprotocols in which such retroviral particles comprising mutant humanfactor IX compositions of the present invention may be used for thetreatment of mammals afflicted with hemophilia

c. Combination Therapy

In addition to therapies based solely on the delivery of the mutanthuman factor IX, combination therapy is specifically contemplated. Inthe context of the present invention, it is contemplated that the mutanthuman factor IX therapy could be used similarly in conjunction withother agents for commonly used for the treatment of hemophilia.

To achieve the appropriate therapeutic outcome, using the methods andcompositions of the present invention, one would generally provide acomposition comprising the mutant human factor IX and at least one othertherapeutic agent (second therapeutic agent). In the present invention,it is contemplated that the second therapeutic agent may be one or moreother factors involved in the blood coagulation cascade. For example, itis contemplated that the compositions comprising the mutant human factorIX of the present invention may be combined with activated prothrombincomplex concentrates, factors II, VII, VIIa, VIII, X, precursor Xa,protein C, XI and XII.

The combination therapy compositions would be provided in a combinedamount effective to produce the desired therapeutic outcome of bloodcoagulation. This process may in-solve contacting the cells with themutant human factor IX and the second agent(s) or factor(s) at the sametime. This may be achieved by administering a single composition orpharmacological formulation that includes both agents, or byadministering two distinct compositions or formulations, at the sametime, wherein one composition includes the mutant human factor IXtherapeutic composition and the other includes the second therapeuticagent.

Alternatively, the mutant human factor IX treatment may precede orfollow the other agent treatment by intervals ranging from minutes toweeks. In embodiments where the second therapeutic agent and the mutanthuman factor IX are administered separately, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the second agent and mutant human factor IXwould still be able to exert an advantageously combined effect. In suchinstances, it is contemplated that one would administer both modalitieswithin about 12–24 hours of each other and, more preferably, withinabout 6–12 hours of each other, with a delay time of only about 12 hoursbeing most preferred. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

Local delivery of mutant human factor IX expression constructs orproteins to patients may be a very efficient method for delivering atherapeutically effective gene to counteract the immediate clinicalmanifestations of the disease, i.e., localized bleeding. Similarly, thesecond therapeutic agent may be directed to a particular, affectedregions of the subject's body. Alternatively, systemic delivery of themutant human factor IX and/or the second therapeutic agent may beappropriate in certain circumstances.

G. Assays for Factor IX Activity

In certain aspects of the present invention, it may be necessary todetermine the activity of mutant human factor A. In particular, theeffect of the therapeutic compositions of the present invention on bloodcoagulation activity may need to be monitored Those of skill in the artare aware of numerous blood coagulation assays some of which aredescribed in the present section. This is by no means intended to be anexhaustive list of such assays and is merely intended to provide certainexemplary assays well known to those of skill in the art that may beused in determining the blood coagulation activity of the presentinvention. Further, the present section also describes assays for thedetermination of heparin inhibition of factor IX activity. Exemplary invitro and in vivo assays for determining these activities are providedherein below.

a. In Vitro Assays

A chromogenic in vitro assay may be used to determine the human factorIX activity of a given mutant human factor IX of the present invention.Factor IXa has poor reactivity toward chromogenic substrates, likely dueto the partially collapsed nature of the active site [Brandstetter etal., Proc Nat'l Acad Sci USA, 92(21): 9796–800 (1995)]. However, theaddition of 30% ethylene glycol can increase the catalytic rate nearlyten fold, especially for substrates with hydrophobic moieties in the P3position [Sturzebecher et al., FEBS Letters, 412(2): 295–300 (1997)].The effect of mutations in the heparin binding site of factor IXa oncleavage of the chromogenic substrates, Pefachrome IXa(CH₃SO₂-D-CHG-Gly-Arg-pNA) or CBS 31.39(CH₃SO₂DLeu-Gly-Arg-p-nitroanilide), can be assessed by incubatingincreasing amounts of heparin with 25 nM enzyme in 0.15 M NaCl, 2 mMCaCl₂, 20 mM HEPES, pH 7.4, 30% ethylene glycol and 2.5 mM PefachromeIXa or 4 mM CBS 31.39 in a microtiter plate. Initial rates aredetermined by the change in absorbance at 405 nm over 5–10 min a VmaxReader. The K_(m) of CBS 31.39 for factor IXa is 3.7 mM under theseconditions.

An in vitro assay for intrinsic complex activity to determine factor IXactivity also may be used. In this assay, thrombin-activated factorVIIIa (final concentration 0.5 nM) is added to a reaction containing 5nM factor IXa, 5% (v/v) rabbit brain cephalin, 300 nM factor X, andincreasing concentrations of heparin in 0.15 M NaCl, 20 mM HEPES, pH7.4, 2 mM CaCl2, and 0.1% PEG-8000. The reaction are sampled (50 μl) at15, 30, 45, and 60 sec into 10 μl of 0.25 M EDTA, pH 8.0. Thechromogenic substrate S-2765 is then added at 300 μM and the amount offactor Xa generated determined by comparison of the rate of cleavagewith a standard curve. The assay for intrinsic tenase activity may bemodified to be performed in the presence of excess factor VIIIa (5 nM)and the linear range for factor IXa determined (as previously describedfor factor VIIIa) for accurate quantitation of the mutant activities.Significant differences in catalytic activity may be further analyzed bydetermination of the Km and kcat for factor X activation by intrinsictenase for wild-type and mutant factor IXa. The affinity of mutantfactor IXa-factor VIIIa complex formation in the presence ofphospholipid can be compared to wild-type factor IXa in a kineticbinding assay.

The relative affinity of heparin for the mutant human factor IXaproteins can be determined by titration of active site-labeled protease.The interaction of heparin with F1-EGR-factor IXa can be detected by thechange in emission fluorescence intensity at 525 nm. To generate theactive site-labeled proteases, wild-type and mutant human factor IX isactivated by incubation with factor XIa. Conditions for completeactivation can be confirmed by SDS-PAGE for each mutant protein. Themutant factor IXa is then incubated with ten-fold molar excess offluorescein-EGR-chloromethylketone (Hematologic Technologies) for 30 minat 23° C., followed by gel filtration chromatography on a G-100 column(fractionation range 4–100 kDa) to separate factor XIa (void volume) andthe low molecular weight free inhibitor from F1-EGR-factor IXa. Thesample may then be subjected to additional dialysis if necessary tocompletely remove free inhibitor. Labeled proteases will then bequantitated by A₂₈₀. The F1-EGR-factor IXa (25 nM) is titrated withsize-fractionated heparin chains to generate binding curves. The bindingcurves for mutant factor IXa can be compared to wild-type underidentical conditions, with fitting to an appropriate site-specificbinding model to provide a KD(obs) [Olson et al., J Biol Chem, 266(10):6342–52 (1991)]. An estimate of the relative affinity of mutant humanfactor IXa for heparin (i.e. rank order) is sufficient to correlate withthe relative effect of mutations on enzymatic activity and inhibition byheparin. This strategy is similar to that used to map the heparinbinding site of thrombin, where NaCl elution from heparin-sepharose wasused as an estimate of heparin affinity, allowing correlation of elutionposition with the rate constant for inhibition by ATIII-heparin [Sheehanand Sadler, Proc Nat'l Acad Sci USA, 91(12): 5518–22 (1994)].

In vitro blood coagulation assays also are well known to those of skillin the art and are described, for example, in Walter et al., [Proc Nat'lAcad Sci USA, 93: 3056–3061 (1996); Hathaway and Goodnight (1993),Laboratory Measurement of Hemostasis and Thrombosis, In: Disorders ofHemostasis and Thrombosis: A Clinical Guide, pp. 21–29)]. These assaysmay be used in the present invention to ensure that the mutant humanfactor IX possesses an appropriate blood coagulation effect. Those ofskill in the art also are referred to “A Laboratory Manual of BloodCoagulation” Austen et al., Blackwell Scientific Publishing (1975) foradditional methods for conducting blood clotting assays.

In preferred embodiments, the effect of mutations in the heparin bindingexosite on the coagulant activity of mutant human factor IX is assessedby performing an activated partial thromboplastin time (APTT) in factorIX deficient plasma [Bajaj et al, Meth Enzymol, 222: 96–128 (1993)]. Therelative coagulant activity of the mutants is determined by comparisonto a standard curve. The APTT reflects both activation of the mutanthuman factor IX by factor XIa, and the enzymatic activity of theprotease in plasma. Unexpected differences can be further analyzed bycomparing mutant human factor IX and factor IXa plasma coagulantactivity of the mutants to wild-type, in order to differentiate effectson activation versus enzymatic activity.

b. In Vivo Assays

Before the mutant human factor IX compositions of the present inventionare employed in human therapeutic protocols, it may be desirable tomonitor the effects of such compositions in animal models. There are anumber of animal models, in vivo assays, previously described by thoseof skill in the art that may be useful in the present invention.

An exemplary animal model for hemophilia B is available. For example, acolony of mice having severe hemophilia B are well known to those ofskill in the art [Lin et al., Blood, 90(10): 3962–6 (1997); Kung et al.,Blood, 91(3): 784–90 (1998); Snyder et al., Nat Med, 5(1): 64–70(1999)]. Additionally, a colony of dogs having severe hemophilia Bcomprising males that are hemizygous and females that are homozygous fora point mutation in the catalytic domain of the canine factor IX gene,have been maintained for more than two decades at the University ofNorth Carolina, Chapel Hill [Evans et al., Blood 74: 207–212 (1989)].

The hemostatic parameters of the above mice and dogs are well described.For example, in the dogs there is an absence of plasma factor IXantigen, whole blood clotting times of>60 minutes, whereas normal dogsare 6–8 minutes, and prolonged activated partial thromboplastin time of50–80 seconds, whereas normal dogs are 13–18 seconds. These dogsexperience recurrent spontaneous hemorrhages. Typically, significantbleeding episodes are successfully managed by the single intravenousinfusion of 10 mL/kg of normal canine plasma; occasionally, repeatinfusions are required to control bleeding.

In order to determine the efficacy of the mutant human factor IX proteinand gene therapy compositions of the present invention, such mice anddogs may be injected intramuscularly and/or intravenously with thecompositions of the present invention and the blood clotting time in thepresence and absence of the compositions may be determined. Suchdeterminations will be helpful in providing guidance on the dosages andtimes of administration and the efficacy of a given composition againsthemophilia B. In gene therapy protocols, immunofluorescence staining ofsections obtained from biopsied muscle may be performed, and expressionof the mutant human factor IX in the transduced muscle fibers may bedetermined.

H. Pharmaceutical Compositions

In order to prepare mutant human factor IX containing compositions forclinical use, it will be necessary to prepare the viral expressionvectors, proteins, and nucleic acids as pharmaceutical compositions,i.e., in a form appropriate for in vivo applications. Generally, thiswill entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the mutant human factor IX or an expression vectorto cells, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The active compositions of the present invention include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. The pharmaceuticalcompositions may be introduced into the subject by any conventionalmethod, e.g., by intravenous, intradermal, intramusclar, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g. termrelease); by oral, sublingual, nasal, anal, vaginal, or transdermaldelivery, or by surgical implantation at a particular site. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time.

The active compounds may be prepared for administration as solutions offree base or pharmacologically acceptable salts in water, suitably mixedwith a surfactant, such as hydroxypropylcellulose. Dispersions also canbe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms, suitable for injectable use, include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion, and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum-drying and freeze-drying techniquesthat yield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as injectable solutions, drug release capsules andthe like. For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered, if necessary, and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration.

“Unit dose” is defined as a discrete amount of a therapeutic compositiondispersed in a suitable carrier. For example, where polypeptides arebeing administered parenterally, the polypeptide compositions aregenerally injected in doses ranging from 1 μg/kg to 100 mg/kg bodyweight/day, preferably at doses ranging from 0.1 mg/kg to about 50 mg/kgbody weight/day. In terms of units of mutant human factor IX activityper kg of weight of subject, it is contemplated that between about 100to about 500 units/kg body weight will be useful. Parenteraladministration may be carried out with an initial bolus followed bycontinuous infusion to maintain therapeutic circulating levels of drugproduct. Those of ordinary skill in the art will readily optimizeeffective dosages and administration regimens as determined by goodmedical practice and the clinical condition of the individual patient.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the routes of administration. The optimal pharmaceuticalformulation will be determined by one of skill in the art depending onthe route of administration and the desired dosage. See, for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publ. Co,Easton Pa. 18042, pp. 1435–1712, incorporated herein by reference. Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface area, or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein, as well as thepharmacokinetic data observed in animals or human clinical trials.

Appropriate dosages may be ascertained through the use of establishedassays for determining blood clotting levels in conjunction withrelevant dose-response data. The final dosage regimen will be determinedby the attending physician, considering factors that modify the actionof drugs, e.g., the drug's specific activity, severity of the damage andthe responsiveness of the patient, the age, condition, body weight, sexand diet of the patient, the severity of any infection, time ofadministration, and other clinical factors. As studies are conducted,further information will emerge regarding appropriate dosage levels andduration of treatment for specific diseases and conditions.

In gene therapy embodiments employing viral delivery, the unit dose maybe calculated in terms of the dose of viral particles beingadministered. Viral doses include a particular number of virus particlesor plaque forming units (pfu). For embodiments involving adenovirus,particular unit doses include 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³ or 10¹⁴ pfu. Particle doses may be somewhat higher (10to 100-fold) due to the presence of infection defective particles.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention may be useful in fields of humanmedicine and veterinary medicine. Thus, the subject to be treated may bea mammal, preferably human or other animal. For veterinary purposes,subjects include, for example, farm animals including cows, sheep, pigs,horses and goats, companion animals, such as dogs and cats, exoticand/or zoo animals, laboratory animals including mice rats, rabbits,guinea pigs and hamsters, and poultry such as chickens, turkeys, ducks,and geese.

I. Examples

The present invention is described in more detail with reference to thefollowing non-limiting examples, which represent preferred embodimentsof the invention. Those of skill in the art will understand that thetechniques described in these examples represent techniques described bythe inventors to function well in the practice of the invention, and assuch constitute preferred modes for the practice thereof. However, itshould be appreciated that those of skill in the art should in light ofthe present disclosure, appreciate that many changes can be made in thespecific methods that are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

EXAMPLE 1 Transient Expression of Human Factor IX

Construction and Transient Expression of Factor IX Constructs. Therecombinant human factor IX cDNA in the expression vector pCMV5 wasgenerously provided by Darrel Stafford (Univ. North Carolina). The EcoRIfragment of the cDNA insert was excised from the pCMW5-human factor DCexpression vector and subcloned into pBluescript SK II for mutagenesis.Mutations are constructed by PCR using a high fidelity DNA polymerase(pfu) followed by Dpn I digestion of the parental plasmid (QuikChangeMutagenesis Kit, Strategene). Following transformation, clonescontaining the desired mutation(s) are selected by DNA sequencing. TheEcoRI fragment is excised from plasmids with the desired mutation(s) andsubcloned back into the pCMV5 expression vector. Proper orientation ofthe constructs for expression is confirmed by restriction digest withBam HI/Bgl II. Mutant factor IX cDNA constructs are screened for proteinexpression and initial characterization as described below.

Initial Characterization of Transiently Expressed Factor IX Proteins.The use of initial characterization of constructs in transienttransfections will allow one of skill in the art to monitor and modifythe results of the mutagenesis strategy to assist in the selection ofstable cell lines and purification of the mutant protein.

The pCMV5-HuFIX wild-type construct was transiently transfected into 293cells with Lipofectin (Gibco-BRL). Following transfection, cells wereincubated in serum free media (SFM) containing 10 μg/mL Vitamin K for 48hr. The SFM was harvested, concentrated 10–12 fold by Centricon-30, andassayed for clotting and intrinsic tenase activity.

Clotting activity and intrinsic tenase activity were easily detected inthe media, with no significant background detected with mock-transfectedcells. Factor IX antigen concentration was determined using a “sandwich”type ELISA with affinity purified sheep anti-human factor IX polyclonalantibody (Hematologic Technologies) as the capture antibody, and ahorseradish peroxidase-conjugated affinity purified sheep anti-humanfactor IX antibody (Enzyme Research) to detect the immobilized antigen.The assay demonstrated a linear relationship (log-log plots) from 0.1 to100 μg/mL human factor IX, and estimated factor IX concentrations in the0.6 μg/mL range (10–12 nM) following transient transfection ofpCMV5-factor IX.

Factor X activation by the mutant proteins may be determined in theintrinsic tenase assay following activation from the zymogen form withfactor XIa. In this method, thrombin-activated factor VIIIa (finalconcentration 0.5 nM) is added to a reaction containing 5 nM factor IXa,5% (v/v) rabbit brain cephalin, 300 nM factor X, and increasingconcentrations of heparin in 0.15 M NaCl, 20 mM HEPES, pH 7.4, 2 mMCaCl₂, and 0.1% PEG-8000. The reaction is sampled (50 μl) at 15, 30, 45,and 60 sec into 10 μl of 0.25 M EDTA, pH 8.0. The chromogenic substrate,S-2765, is then added at 300 μM and the amount of factor Xa generateddetermined by comparison of the rate of cleavage with a standard curve[Sheehan and Lan, Blood, 92(5): 1617–1625 (1998)]. Alternatively, thischromogenic assay for factor Xa generation can be made more quantitativefor factor IXa (0.5 nM) by performing it in the presence of excessfactor VIIIa (5 nM).

Coagulant activity of the mutant proteins is determined by an APTT infactor IX deficient plasma, with comparison to a standard curve [Bajajet al., Meth Enzymol, 222: 96–128 (1993)].

EXAMPLE 2 Stable Expression and Purification of Human Factor IX

The present example provides methods for the recombinant expression andpurification of recombinant human factor IX. Stable cell linesexpressing recombinant factor IX are selected for large scale productionof protein, and factor IX is purified to homogeneity from serum-freeconditioned media.

Stable cell lines expressing recombinant human factor IX were obtainedby co-transfecting ScaI-linearized pCMV5-huFIX and pSV2neo plasmids into293 cells by the calcium phosphate method, and selecting clones bylimiting dilution in G418. Cell lines expressing high levels of therecombinant factor IX were determined by ELISA and/or Western blot. Celllines with the highest expression levels were then expanded forlarge-scale culture in T-225 cm² flasks. Upon reaching confluence, thegrowth media (50% DME/50% F-12/10% FCS) was removed, the monolayerswashed extensively with SFM, and replaced with SFM supplemented withinsulin-transferrin-sodium selenite (Sigma) and 10 μg/mL vitamin K.Conditioned media was collected every 48 hours for 10 days. Benzamidinewas added to a final concentration of 5 mM, and the conditioned mediafrozen at −20° C. to −25° C. Upon thawing, the conditioned media waspooled, filtered, and subjected to barium chloride precipitation [Coteet al., J Biol Chem, 269(15):11374–80 (1994)]. The precipitate wasdissolved in 0.2 M EDTA, and the eluate dialyzed overnight beforeapplication to a Mono Q HR 5/5 column (0.15 M NaCl, 20 mM HEPES, pH 7.4,0.1% PEG-8000). Human factor IX was eluted with a calcium chloridegradient (0–45 mM) and concentrated in a Centricon-30.

Purity of factor IX proteins was assessed by SDS-PAGE with silverstaining, and the factor IX concentration was determined by A280(1%=13.3) and ELISA. The human factor IX isolated above demonstratedhigh purity by 10% SDS-PAGE with silver staining (FIG. 1), high specificclotting activity (187 U/mg), and an overall yield of approximately 30%by ELISA. Additionally, elimination of factor IX antigen with lowclotting activity (partial degradation or incomplete carboxylation) wasdetected by Western blot following 2 M NaCl elution of the Mono Q columnfollowing the calcium chloride gradient.

Other studies maybe used to determine the activity of the recombinantfactor IXa. Recombinant factor IX may be activated by incubation withhuman factor XIa (Enzyme Research) at a molar ratio of 100:1 in 150 mMNaCl, 20 mM HEPES, 2 mM CaCl2, pH 7.4. for 2 hr at 37° C. Activation ismonitored by SDS-PAGE, and factor IXa specific activity estimated byactive site titration with antithrombin [Chang et al., J Biol Chem,273(20): 12089–94 (1998)].

Using the methods described in the present example, it is possible topurify highly active recombinant factor IX to homogeneity for detailedanalysis of enzymatic and binding properties.

EXAMPLE 3 Three-Dimensional Model of Factor IX

A three-dimensional structure of human factor IXa was obtained byhomology modeling with SWISS-MODEL, using the crystal structures ofrecombinant human factor IXa complexed with p-aminobenzamidine (1RFN),porcine factor IXa complexed with D-FPR-chloromethylketone (1PFX), humanfactor VIIa with soluble tissue factor (1DAN), and human factor Xacomplexed with the synthetic inhibitor FX-2212A (1XKA, 1XKB) astemplates [Hopfner et al., Structure Fold Des, 7(8): 989–96 (1999);Brandstetter et al., Proc Nat'l Acad Sci USA, 92(21): 9796–800 (1995);Banner et al., Nature, 380(6569): 41–6 (1996)]. The availability of athree-dimensional model of the protease is extremely helpful forplanning and modification of the mutagenesis strategy.

EXAMPLE 4 Expression of Mutant Human Factor IX

The factor IX R233A construct was transiently expressed in 293 cells,concentrated by Centricon-30, and tested for clotting and intrinsictenase activity. Initial experiments demonstrated roughly equivalentclotting activity to wild-type factor IX in factor IX-deficient plasma.After activation with factor XIa in conditioned media, the inhibitoryeffects of heparin on intrinsic tenase activity were tested in thepresence of excess factor VIIIa. The relationship between clottingactivity and intrinsic tenase activity in the absence of inhibitors wasroughly proportionate for both recombinant proteases. Compared towild-type factor IXa, the mutant R233A demonstrated markedly reducedinhibition by heparin.

Although a KJ cannot be calculated from the transient transfection data,a marked increase in the residual activity in the plateau phase wasnoted for the mutant R233A (˜65%) relative to wild-type factor IXa(˜15%) (FIG. 2). Similar effects on heparin inhibition were noted intransient transfection experiments with the factor IX K241A construct.

EXAMPLE 5 Comparison of In Vitro Antithrombin-Independent Inhibition ofWild-Type and Mutant R-233A Human Factor IXa by Unfractionated Heparin

The present example demonstrates the resistance of the purified factorIXa mutant R233A to antithrombin independent inhibition byunfractionated heparin. Factor Xa generation by 5 nM wild-type (●) orR233A (∘) factor IXa in the intrinsic tenase complex (0.5 nM factorVIIIa. 5% rabbit brain cephalin, 300 nM factor X and 2 mM CaCl₂) wasdetermined in the presence of increasing amounts of unfractionatedheparin (as described in Example 1; also, see FIG. 4). The data were fitby nonlinear regression to the equation for partial, uncompetitiveinhibition. The mutant factor IXa R233A demonstrates increasedresistance to inhibition by heparin, as demonstrated by the significantreduction in maximal inhibition (increase in enzymatic activity)observed relative to wild-type factor IXa.

EXAMPLE 6 Kinetic Analysis of Activation by Human Factor XIa onWild-Type and Recombinant Human Factor IXa Mutant Proteins

293 cells were co-transfected with pSV2neo and pCMV5-huFIX constructs,and stable cell-lines expressing the recombinant human factor IXproteins were selected by resistance to the antibiotic G418. Humanfactor A. H92A, R233A, and K241A were purified to homogeneity fromconditioned media. Clotting activity was determined in an APTT assayperformed in factor IX deficient plasma (Table D). Wild-type and factorIX R233A were activated to factor IXa with human factor XIa. Analysis ofthe time course for factor IX activation by human factor XIa on an 10%SDS-PAGE gel demonstrated no significant difference between wild-typefactor IX and the mutant R233A.

Following activation to factor IXa, the ability of heparin to inhibitfactor X activation by the recombinant proteins was examined for boththe factor IXa-phospholipid (in the presence of 30% ethylene glycol) andintrinsic tenase complex (factor VIIIa-factor IXa-phospholipid) (FIGS.4A and 4B). Factor IXa R233A demonstrated resistance to inhibition byheparin relative to wild-type factor IXa under both assay conditions,suggesting that this mutation adversely affected the interaction offactor IXa with heparin.

TABLE D Relative Heparin Affinity and Clotting Activity of RecombinantFactor IX Mutant Proteins. Heparin-Sepharose Clotting Activity ProteinElution (M NaCl) (% normal) Plasma-derived factor 0.49 N.D. IXPlasma-derived factor 0.58 100% IXa Wild-type factor IXa 0.58  90%Factor IXa H92A 0.54  93% Factor IXa R170A 0.56 N.D. Factor IXa R233A0.33  36% Factor IXa K241A 0.56  55% Relative heparin affinity wasdetermined in purified plasma-derived or recombinant factor IX. Controlor mutant protein (30 μg) wasactivated with 25 nM human factor XIa(200:1, substrate:enzyme) for 2 hr at 37° C. and then applied to aheparin-sepharose column(1 mL) at a flow rate of 0.5 mL/min. The columnwas washed with 10 mL of 0.05 M NaCl, 20 mM HEPES, pH 7.4, 0.1%PEG-8000, and 5 mM EDTA,followed by elution with a 0.05 to 1.0 M NaClgradient at 1 mL/min. Clotting activity was determined in an APTT assayperformed byaddition of the zymogen from (factor IX) to factor IXdeficient plasma (with comparison to a standard curve).

EXAMPLE 7 Heparin Binding Affinity of Wild-Type and Recombinant HumanFactor IXa Mutant Proteins

The relative affinity of the recombinant proteins for heparin wasassessed by the position of elution from a heparin-sepharose column inresponse to a NaCl gradient. Heparin-protein interactions are generallydominated by electrostatic forces, thus, this assay is a reasonablesurrogate for direct binding assays. The interaction of the homologouscoagulation protease thrombin with heparin has been examined in detail,demonstrating the predominant contribution of electrostatic forces[Olson et al, J Biol Chem, 266(10): 6342–52 (1991)]. Likewise, theeffect of mutations on the affinity of recombinant thrombins for heparinhas previously been assessed by NaCl elution from heparin-sepharose, andcorrelated with the functional effects of these mutations on inhibitionby antithrombin-heparin [Sheehan et al., Proc Nat'l Acad Sci USA;91(12): 5518–22 (1994)].

Substitution of alanine for homologous residues in human factor IXaresulted in an elution of the recombinant factor IXa from the heparinsepharose at a lower concentration of NaCl, consistent with a reductionin heparin affinity.

Plasma-derived factor IX and IXa eluted from heparin-sepharose at 0.49and 0.58 M NaCl, respectively, suggesting that the activated proteasebinds with higher affinity than the zymogen form. Wild-type factor IXaeluted similarly to plasma-derived factor IXa, suggesting that anydifferences in post-translational modifications between these proteinsdid not affect heparin binding. The remainder of the recombinant factorIXa proteins demonstrated either modest or marked reduction in apparentheparin affinity (elution at lower NaCl concentration). The first groupincluded factor IXa H92A, R170A, and K241A, which demonstrated modestreductions in apparent heparin affinity. The second group includedfactor IXa IR233A, which demonstrated a marked reduction in apparentheparin affinity (Table D). These data demonstrate that the selectedmutations (especially R233A) reduce apparent heparin affinity,suggesting that these residues contribute to a heparin-binding exositeon factor IXa.

The modest effect of single alanine substitutions is not unexpectedgiven the electrostatic, multivalent nature of heparin-protease binding[Olson et al., supra (1991)], and either charge reversal (substitutionof glutamate/aspartate) or combinatorial mutants are expected tosignificantly enhance this effect. The relative effects of thesemutations on apparent heparin affinity of factor IXa suggest that theheparin-binding exosite maps to the carboxyl-terminus region of theprotease.

EXAMPLE 8 Clotting Activity of Wild-Type and Recombinant Human FactorIXa Mutant Proteins

Clotting activity was examined in a modified activated partialthromboplastin time for both plasma-derived and recombinant factor IXa.Consistent with previous reports, wild-type factor IXa demonstratedapproximately 90% of plasma-derived factor IXa clotting activity. Thisresult may be secondary to the presence of a minor factor IX form (4–5%)in which the prosequence has not been cleaved [Bajaj et al., J BiolChem, 272(37): 23418–26 (1997)]. Thus, the wild-type factor IXa clottingactivity represents the appropriate control for comparison of therecombinant factor IXa mutant proteins. Factor IXa R233A and K241Ademonstrated moderate reductions in clotting activity relative towild-type, while factor IXa H92A had similar clotting activity towild-type factor IXa. Likewise, factor IXa R170A was reported to haveincreased clotting activity relative to wild-type or plasma-derivedfactor IXa [Chang et al., J Biol Chem, 273(20): 12089–94 (1998)]. Thus,the effect of amino acid substitutions on relative heparin affinity canclearly be dissociated from effects on clotting activity.

While the methods and compositions herein have been described in termsof preferred embodiments, it will be apparent that variations may beapplied to the methods and/or compositions without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that assays that are physiologically related may besubstituted for the assays described herein while still producing thesame or similar results. All such similar substitutes and modificationsapparent to those of skill in the art are deemed to be within the scopeof the invention as defined by the appended claims.

The present specification cites to certain scientific journal referencesand patents that, to the extent that they provide exemplary proceduralor other information supplemental to that set forth herein, arespecifically incorporated herein by reference.

1. A mutant human factor IX comprising a mutation in the heparin bindingdomain which decreases its affinity for heparin as compared to wild-typehuman factor IX, wherein said mutation is a mutation of the amino acidresidue 298, 303, 304, 312, 315, 447, 450, 456, or 458 of SEQ ID NO: 2.2. The mutant human factor IX of claim 1, wherein said mutation furthercomprises a substitution of arginine for an alanine at amino acidresidue 385 of SEQ ID NO:
 2. 3. A mutant human factor IX having amutation at amino acid residue 450 of SEQ ID NO: 2, wherein saidmutation decreases the affinity of said mutant human factor IX forheparin as compared to wild-type human factor IX.
 4. The mutant humanfactor IX of claim 3, wherein said mutation is a substitution of thearginine at amino acid residue 450 of SEQ ID NO: 2 to any other aminoacid.
 5. The mutant human factor IX of claim 4, wherein arginine atamino acid residue 450 of SEQ ID NO: 2 is substituted with an alanine.6. A method of treating a subject having hemophilia comprisingadministering to said subject a composition comprising a mutant humanfactor IX of claim 1, in an amount effective to promote blood clottingin said subject.
 7. The method of claim 6, wherein said mutant humanfactor IX comprises a mutation of the amino acid located at residue 450of SEQ ID NO:2.
 8. The method of claim 7, wherein the mutant humanfactor IX comprises a mutation of arginine to an alanine at amino acidresidue 450 of SEQ ID NO:2.
 9. The method of claim 6, further comprisingadministering to said subject a composition comprising one or moreadditional blood clotting factors other than said mutant human factorIX.
 10. The method of claim 6, wherein said hemophilia is hemophilia B.11. A pharmaceutical composition comprising the mutant human factor IXof any one of claims 1 through 5 and a pharmaceutically acceptablecarrier, excipient, or diluent.